Unnatural amino acids represent one of the largest and growing classes of intermediates used by the pharmaceutical, agrochemical and fine chemical industries. Demand for these compounds at large scale and high optical purity is now very significant, due to their application and success in single enantiomer pharmaceuticals, particularly for the areas of virology and oncology. No synthetic or biosynthetic method however, has proven sufficiently versatile to prepare these compounds broadly at scale. Richmond Chemical Corporation (RC Corp.) has recently developed an efficient and general chemo-enzymatic process to prepare enantiomerically pure L- and D-amino acids in high yield by deracemization of racemic starting materials. This method involves the concerted action of an enantioselective amino acid oxidase biocatalyst and a non-selective chemical reducing catalyst to affect the stereo-inversion of one enantiomer, and can result in an enantiomeric excess (ee) of >99 % from the starting racemate, with product yields of over 90 %. This approach, conducted entirely in water, (thereby minimizing waste streams) compares very favorably to resolution processes, which have a maximum single pass yield of 50 %. We have successfully developed methods to optimize the efficiency of the process and establish competitive economics at scale. However, the breadth of application of this process is limited by the substrate range of the native oxidase biocatalysts. In the Phase I work of this project, we successfully applied powerful methods of in vitro enzyme evolution to demonstrate the adaptability of the oxidase biocatalysts, by successfully isolating variants of D-amino acid oxidase with high activity and enantioselectivity towards sterically bulky amino acids of high commercial importance. These amino acids are of high relevance due to their favorable pharmacokinetic properties but are difficult to prepare by traditional methods. In addition, the stability of the natural D-amino acid oxidase was greatly enhanced using the same approach. In parallel we successfully identified conditions in which the reduction step can be carried out with near quantitative yield under mild conditions. These results validate key aspects required for deracemization as a platform bioprocess and we now propose in Phase II to develop the scalable chemo-enzymatic deracemization processes to prepare these and additional unnatural amino acids for industry. We will continue the directed evolution of the oxidase biocatalyst and optimize critical reaction parameters including substrate load, biocatalyst production, formulation and re-use, as well as product isolation and recovery. The optimized deracemization processes will be implemented in the commercial manufacture of unnatural amino acids at large-scale. This work plan will result in new general bioprocess technology for the fine chemical industry, to produce chiral synthetic intermediates for pharmaceutical applications at commercial scale. PUBLIC HEALTH RELEVANCE: This project is aimed towards the development of an economical enzymatic process for the synthesis of optically pure unnatural amino acids. These amino acids are in great demand for the synthesis of a wide range of important pharmaceutical compounds such HIV protease inhibitors, anti-cancer agents and anti-diabetic drugs.